Ye Shaojing, Agalave Nilesh M, Ma Fei, Mahmood Dlovan F D, Al-Grety Asma, Khoonsari Payam E, Leng Lin, Svensson Camilla I, Bucala Richard, Kultima Kim, Vera Pedro L
Research & Development, Lexington VA Health Care System, Lexington, KY 40502, USA.
Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85 Uppsala, Sweden.
Int J Mol Sci. 2024 Apr 19;25(8):4484. doi: 10.3390/ijms25084484.
Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 μg); or MIF mAb (15 μg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.
膀胱疼痛是间质性膀胱炎/膀胱疼痛综合征（IC/BPS）的一个突出症状。我们使用一种模型研究了小鼠膀胱疼痛的脊髓机制，在该模型中，膀胱内蛋白酶激活受体-4（PAR4）的反复激活会导致持续性膀胱痛觉过敏（BHA），且几乎没有膀胱炎症。持续性BHA由脊髓巨噬细胞移动抑制因子（MIF）介导，并与腰骶部蛋白质组学的变化有关。我们研究了单个脊髓MIF受体对持续性膀胱疼痛的作用，以及与脊髓MIF拮抗缓解持续性BHA相关的脊髓蛋白质组学变化。患有持续性BHA的雌性小鼠接受鞘内注射（i.t.）MIF单克隆抗体（mAb）或小鼠IgG1（同型对照抗体）。MIF拮抗作用暂时逆转了持续性BHA（峰值效应：2小时），而对照IgG1没有效果。此外，鞘内注射MIF受体CD74和C-X-C趋化因子受体4型（CXCR4）的拮抗剂可部分逆转持续性BHA。对于蛋白质组学实验，四组不同的小鼠分别接受反复膀胱内注射乱序肽和假鞘内注射（对照组，无疼痛组）或反复膀胱内注射PAR4和：假鞘内注射；同型IgG1鞘内注射（15μg）；或MIF mAb（15μg）。注射后2小时切除L6-S1脊髓节段，使用液相色谱-串联质谱（LC-MS/MS）检测蛋白质组学变化。无偏蛋白质组学分析鉴定并相对定量了6739种蛋白质。我们选择了在膀胱内注射PAR4（假鞘内注射或IgG鞘内注射治疗）后与对照组（无疼痛组）相比有显著变化，且在鞘内注射MIF拮抗剂后无显著变化的蛋白质。在持续性BHA期间，有6种蛋白质减少（免疫组织化学证实为含V-set跨膜结构域蛋白2样），而有2种蛋白质增加。脊髓MIF拮抗作用逆转了蛋白质变化。因此，脊髓MIF和MIF受体介导持续性BHA以及特定脊髓蛋白质的变化。这些新的MIF调节的脊髓蛋白质代表了可能破坏介导持续性膀胱疼痛的脊髓机制的新靶点。
